scholarly journals Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog

2019 ◽  
Author(s):  
Yun Huang ◽  
Xiaoyu Wang ◽  
Guohua Lv ◽  
Asghar M. Razavi ◽  
Gerard H. M. Huysmans ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Membrane Proteins ◽  
Binding Site ◽  
Conformational Changes ◽  
Glutamate Transporter ◽  
Conformational Exchange ◽  
Substrate Binding Site ◽  
Domain Movement ◽  
Paramagnetic Metal ◽  
Longitudinal Relaxation

AbstractIn proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homologue with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified two outward- and one inward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by Cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

scholarly journals Structural Mechanism of Transport of Mitochondrial Carriers

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
J. J. Ruprecht ◽  
E.R.S. Kunji
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Substrate Binding ◽  
Ion Homeostasis ◽  
Annual Review ◽  
Publication Date ◽  
Intermembrane Space ◽  
Substrate Binding Site ◽  
Mitochondrial Carriers ◽  
The Matrix

Members of the mitochondrial carrier family [solute carrier family 25 (SLC25)] transport nucleotides, amino acids, carboxylic acids, fatty acids, inorganic ions, and vitamins across the mitochondrial inner membrane. They are important for many cellular processes, such as oxidative phosphorylation of lipids and sugars, amino acid metabolism, macromolecular synthesis, ion homeostasis, cellular regulation, and differentiation. Here, we describe the functional elements of the transport mechanism of mitochondrial carriers, consisting of one central substrate-binding site and two gates with salt-bridge networks on either side of the carrier. Binding of the substrate during import causes three gate elements to rotate inward, forming the cytoplasmic network and closing access to the substrate-binding site from the intermembrane space. Simultaneously, three core elements rock outward, disrupting the matrix network and opening the substrate-binding site to the matrix side of the membrane. During export, substrate binding triggers conformational changes involving the same elements but operating in reverse. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Activation of the Redox-regulated Chaperone Hsp33 by Domain Unfolding

2004 ◽  
Vol 279 (19) ◽  
pp. 20529-20538 ◽  
Author(s):  
Paul C. F. Graf ◽  
Maria Martinez-Yamout ◽  
Stephen VanHaerents ◽  
Hauke Lilie ◽  
H. Jane Dyson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Binding Site ◽  
Conformational Changes ◽  
Substrate Binding ◽  
Helical Structure ◽  
Activation Process ◽  
Spectroscopic Techniques ◽  
Substrate Binding Site ◽  
C Terminus ◽  
Dimerization Interface

The molecular chaperone Hsp33 inEscherichia coliresponds to oxidative stress conditions with the rapid activation of its chaperone function. On its activation pathway, Hsp33 progresses through three major conformations, starting as a reduced, zinc-bound inactive monomer, proceeding through an oxidized zinc-free monomer, and ending as a fully active oxidized dimer. While it is known that Hsp33 senses oxidative stress through its C-terminal four-cysteine zinc center, the nature of the conformational changes in Hsp33 that must take place to accommodate this activation process is largely unknown. To investigate these conformational rearrangements, we constructed constitutively monomeric Hsp33 variants as well as fragments consisting of the redox regulatory C-terminal domain of Hsp33. These proteins were studied by a combination of biochemical and NMR spectroscopic techniques. We found that in the reduced, monomeric conformation, zinc binding stabilizes the C terminus of Hsp33 in a highly compact, α-helical structure. This appears to conceal both the substrate-binding site as well as the dimerization interface. Zinc release without formation of the two native disulfide bonds causes the partial unfolding of the C terminus of Hsp33. This is sufficient to unmask the substrate-binding site, but not the dimerization interface, rendering reduced zinc-free Hsp33 partially active yet monomeric. Critical for the dimerization is disulfide bond formation, which causes the further unfolding of the C terminus of Hsp3 and allows the association of two oxidized Hsp33 monomers. This then leads to the formation of active Hsp33 dimers, which are capable of protecting cells against the severe consequences of oxidative heat stress.

scholarly journals Selective Interface Detection:  Mapping Binding Site Contacts in Membrane Proteins by NMR Spectroscopy

2005 ◽  
Vol 127 (16) ◽  
pp. 5734-5735 ◽  
Author(s):  
Suzanne R. Kiihne ◽  
Alain F. L. Creemers ◽  
Willem J. de Grip ◽  
Petra H. M. Bovee-Geurts ◽  
Johan Lugtenburg ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Membrane Proteins ◽  
Binding Site

The Structural Landscape of SARS-CoV-2 Main Protease: Hints for Inhibitor Search.

2020 ◽  
Author(s):  
Luigi Leonardo Palese
Keyword(s):  
Comparative Analysis ◽  
Global Health ◽  
Binding Site ◽  
Causative Agent ◽  
Data Set ◽  
Substrate Binding Site ◽  
Global Pandemic ◽  
Main Protease ◽  
Crystallographic Structures ◽  
Structural Aspects

In 2019, an outbreak occurred which resulted in a global pandemic. The causative agent of this serious global health threat was a coronavirus similar to the agent of SARS, referred to as SARS-CoV-2. In this work an analysis of the available structures of the SARS-CoV-2 main protease has been performed. From a data set of crystallographic structures the dynamics of the protease has been obtained. Furthermore, a comparative analysis of the structures of SARS-CoV-2 with those of the main protease of the coronavirus responsible of SARS (SARS-CoV) was carried out. The results of these studies suggest that, although main proteases of SARS-CoV and SARS-CoV-2 are similar at the backbone level, some plasticity at the substrate binding site can be observed. The consequences of these structural aspects on the search for effective inhibitors of these enzymes are discussed, with a focus on already known compounds. The results obtained show that compounds containing an oxirane ring could be considered as inhibitors of the main protease of SARS-CoV-2.

Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules

2020 ◽  
Vol 21 (2) ◽  
pp. 117-130 ◽  
Author(s):  
Mohammad J. Hosen ◽  
Mahmudul Hasan ◽  
Sourav Chakraborty ◽  
Ruhshan A. Abir ◽  
Abdullah Zubaer ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Binding Site ◽  
Glucose Transporter ◽  
Substrate Binding ◽  
Mutation Screening ◽  
Homology Model ◽  
Connective Tissue Disorder ◽  
Crystallographic Structure ◽  
Substrate Binding Site

Objectives: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site. Methods: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed. Results and Discussion: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region. Conclusion: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS.

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